Inflammation and/or infection in amniotic fluid is a risk factor for preterm birth and adverse neonatal outcome, which is present in about 50% of patients with preterm premature rupture of membranes. The prediction or diagnosis of infection/inflammation in amniotic fluid can be performed by microbial culture and amniotic fluid analysis, such as measuring inflammatory markers (cytokine concentration, matrix metal loproteinase enzyme concentration, white blood cell count, etc.). The amniotic fluid analysis method is the most sensitive and specific method for predicting the possibility of preterm birth and assessing the risk for neonatal complications. However, collecting amniotic fluid for analysis requires abdominal amniocentesis, which is an invasive procedure. In particular, as it is often difficult to perform amniocentesis when the volume of amniotic fluid is reduced in patients with premature rupture of membranes, therefore only ⅔ of patients undergo this procedure in US.
Although it is preferable to use amniotic fluid for assessing the inflammation in amniotic fluid, an alternative method using vaginal secretion for predicting inflammation or infection is suggested, because it is an invasive procedure. However, as there is a higher chance of infection since the vaginal fluid is exposed to outside, it is difficult to use vaginal fluid for prediction of inflammation or infection in amniotic fluid. It has also been confirmed that there is a significant difference in the absolute amount of inflammatory markers when compared with amniotic fluid. It has been confirmed that vaginal fluid can not have the predictability as much as the invasively collected amniotic fluid can.
In previous research, the present inventors proved a strong correlation between the concentrations of interleukin-6 (IL-6) in cervical secretion collected by Dacron polyester swab and those in the amniotic fluid collected by abdominal amniocentesis from patients diagnosed with premature rupture of membranes (Jun J K, Yoon B H et al., Am J Obstet Gynecol 183:868-873, 2000). However, as there is a significant difference between the absolute concentrations of two subject samples, it is difficult to apply the cut-off value of IL-6 in the amniotic fluid collected by abdominal amniocentesis to cervical secretion for analysis of inflammation or infection in amniotic fluid (see FIG. 1). Similar results were reported from other investigators demonstrating that the concentration of IL-6 in the amniotic fluid (see FIG. 2a) was lower than that of the IL-6 concentration in cervical secretion (see FIG. 2b) (Rizzo G et al., Gynecol Obstet Invest 1998; 46: 91-5)(see FIG. 2). In addition, the previous research showed that the median value or average value of inflammatory cytokine in cervical secretion was generally measured in pg/ml units, while the median value or average value of cytokine in amniotic fluid was measured in ng/ml units. As the concentration of inflammatory cytokine in cervical secretion is extremely lower than those in amniotic fluid, it is difficult to directly use the concentration of cytokine in cervical fluid to predict the concentration of cytokine in amniotic fluid.
Also, in the previous study of the present inventors on counting white blood cell for measuring the level of inflammation in amniotic fluid, the present inventors confirmed a significantly higher number of white blood cells in amniotic fluid leaked through the cervix than that of white blood cells in amniotic fluid collected by abdominal amniocentesis, suggesting that white blood cell count in amniotic fluid leaked through the cervix was not suitable to be used as a marker for predicting inflammation or infection (see FIG. 3 and Table 1). Matrix metal loproteinase-8 (MMP-8) is known to be the best marker for predicting the level of inflammation and the development of preterm birth and neonatal complications. However, it is difficult to use MMP-8 in fluid leaked through the cervix as a prediction marker for inflammation or infection in amniotic fluid, since in most cases, there was a significant increase in the concentration in amniotic fluid leaked through the cervix, even though the concentration in amniotic fluid collected by abdominal amniocentesis was low (See FIG. 10 and Table 8). On the contrary, IL-10 which is used for determining the level of inflammation in amniotic level was not suitable to be used as a marker for predicting inflammation or infection in amniotic fluid, because IL-10 concentration in amniotic fluid leaked through the cervix decreased significantly, even though the concentration was high in amniotic fluid collected by abdominal amniocentesis (See FIG. 9 and Table 7).
Accordingly, the level of inflammatory markers in vaginal secretion was measured to determine the inflammation or infection in amniotic fluid noninvasively. However, finding a noninvasive method for prediction of inflammation or infection in amniotic fluid was not easy, since there were significant differences in absolute values between inflammatory markers in vaginal secretion and those in amniotic fluid. Therefore, it is important to prove the correlation between inflammatory markers in amniotic fluid collected from patients with premature rupture of membranes by abdominal amniocentesis and those in the amniotic fluid leaked through the cervix into the vagina, and furthermore to prove similarities in their absolute concentrations. These will provide a significant improvement in prediction or diagnosis of infection and/or inflammation in amniotic fluid by analyzing the amniotic fluid leaked through the cervix without performing invasive abdominal amniocentesis in patients with premature rupture of membranes.
TABLE 1Sensitivity and specificity for predicting infect ion/inflammationin amniotic fluid according to each of the cut-off values of white cellcount in the amniotic fluid leaked through the cervix into the vagina.Cutt-off value (cells/mm2)SensitivitySpecificity10090.9% (10/11)26.7% (6/15)50072.7% (8/11)53.3% (8/15)100054.5% (6/11)66.7% (10/15)
Until now, there has been no investigation on noninvasive prediction of infection/inflammation in the uterus by comparing the absolute values and by determining correlation of various markers between amniotic fluid collected from patients with premature rupture of membranes by abdominal amniocentesis and amniotic fluid leaked through the cervix into the vagina. Instead of using special equipment to collect amniotic fluid from posterior fornix, most of the previous studies used basic equipment such as bags or vacuum bulb. This caused the increased risk of contamination by bacteria in the vagina, making amniotic fluid from posterior fornix difficult to be used in prediction of infection/inflammation in the uterus. Therefore, in most studies, the amniotic fluid leaked through the cervix into the vagina was used for only determining the fetal pulmonary maturity. Most of the recent studies have focused on confirming the usefulness of phosphatidylglycerol (PG) in predicting the fetal pulmonary maturity from amniotic fluid collected from patients with premature rupture of membranes (Stedman C M et al., Am J Obstet Gynecol 140:34-38, 1981).
The present inventors have measured the concentration of cytokines including IL-6 (Interleukin-6), IL-1 (Interleukin-1), IL-8 (Interleukin-8), monocyte chemo-attractant protein-1 (MCP-1) and Growth Related Oncogene-α (GRO-α) as inflammatory markers. As a result, the present inventors indentified a correlation in the amount of inflammatory markers between the amniotic fluid collected from patients with premature rupture of membranes by abdominal amniocentesis and noninvasively collected amniotic fluid leaked through the cervix into the vagina. The usefulness of 5 cytokines to predict inflammation or infection in amniotic fluid was also confirmed on the basis of the similarity in order of concentration. The present inventors confirmed that these cytokines in amniotic fluid leaked through the cervix into the vagina can be effectively used as inflammatory markers for predicting or diagnosis of inflammation/infect ion in amniotic fluid, thereby leading to the completion of the present invention.